Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro

The ribosome is a macromolecular machine that catalyzes the sequence-defined polymerization of L-α-amino acids into polypeptides. The catalysis of peptide bond formation between amino acid substrates is based on entropy trapping, wherein the adjacency of transfer RNA (tRNA)-coupled acyl bonds in the P-site and the α-amino groups in the A-site aligns the substrates for coupling. The plasticity of this catalytic mechanism has been observed in both remnants of the evolution of the genetic code and modern efforts to reprogram the genetic code (e.g., ribosomal incorporation of non-canonical amino acids, ribosomal ester formation). However, the limits of ribosome-mediated polymerization are underexplored. Here, rather than peptide bonds, we demonstrate ribosome-mediated polymerization of pyridazinone bonds via a cyclocondensation reaction between activated γ-keto and α-hydrazino ester monomers. In addition, we demonstrate the ribosome-catalyzed synthesis of peptide-hybrid oligomers composed of multiple sequence-defined alternating pyridazinone linkages. Our results highlight the plasticity of the ribosome’s ancient bond-formation mechanism, expand the range of non-canonical polymeric backbones that can be synthesized by the ribosome, and open the door to new applications in synthetic biology.


Materials and Methods
Mass spectra were recorded on a Bruker Rapiflex, Bruker Autoflex, AmaZon SL, or Waters Q-TOF Ultima for electron-spray ionization (ESI) and Impact-II or Waters 70-VSE for electron impact (EI).
High resolution mass spectrometry (HRMS) analysis was performed by the University of Texas, Pohang To a biphasic mixture of L-alanine-DBE TFA (132 mg, 346 µmol, 1.00 equiv.) in THF (2.5 mL) and satd. NaHCO3 (aq) (2.5 mL) was added oxaziridine (100 mg, 346 µmol, 1.00 equiv.) dropwise. The reaction was allowed to stir for 120 min under ambient conditions before the reaction mixture was extracted three times with 20 mL of EtOAc. The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to yield an oil. The product was purified by silica gel flash chromatography (35% EtOAc/Hexanes) to yield the product as a

1) N-terminal incorporation
As a reporter peptide, a T7 promoter-controlled DNA template (pJL1_MT_StrepII) was designed to encode a streptavidin (Strep) tag and additional Met (AUG-X) and Thr (ACC-Y) codons (XYWHSPQFEK). The initiation codon AUG and ACC were used for N-terminal incorporation of the γ-keto and hydrazineyl ester substrates, respectively). The PURExpress TM Δ (aa, tRNA) kit (NEB, E6840S) was used for pyridazinone formation reaction and the reaction was performed with only the 8 amino acids that decode the purification tag. The reaction mixtures were incubated at 37 °C for 2 h. The synthesized peptides were then purified using Strep-Tactin®-coated magnetic beads (IBA) and characterized by MALDI-TOF mass spectrometry.

2) C-terminal incorporation (alternating consecutive incorporation)
For alternating incorporations at the C-terminal region of a peptide, the pJL1-StrepII_TI2 and pJL1-StrepII_TI3 encoding the same amino acids (MWHSPQFEKSXYXY or MWHSPQFEKSXYXYXY), where X (Thr:ACC) and Y (Ile:AUC) indicate the position of the γ-keto amino acid (7) and (S)-HzAla (6) substrates, respectively. The reaction condition, purification and characterization methods are the same with the methods described in the paragraph above.

3) Effect of other translational machinery for pyridazinone bond formation
For this study, a custom-made PURExpress® Δ (aa, tRNA, ribosome) kit (NEB, E3315Z) and the wildtype ribosome provided in the kit was not used. To investigate the engineered ribosome's effect, 15 μM (final concentration) of the engineered ribosome (Hecht's 040329) 5 was added to the reaction mixture that contains the 8 amino acids decoding the strep-tag. To investigate the EF-P's effect, additional 10 μM of EF-P 6 was added into the reaction mixture. The reaction condition, purification, and characterization methods are the same with the methods described in the paragraph above.

Preparation of standard peptides
The standard peptides were prepared using PURExpress TM (NEB, E6800S) in the presence of the 20 natural amino acids and the pJL1_MT_StrepII plasmid that decodes the fMetThrTrpHisSerProGlnPheGluLys

Supplementary Figures
Supplementary Figure 1. Acylation of microhelix with substrates 1-7. The Fx-catalyzed acylation reaction using the 7 substrates were monitored at two different pH (7.5 and 8.8) over 48 h with three different flexizymes (eFx, dFx, and aFx). The yield of each reaction was determined by quantifying the relative band intensity of unacylated and acylated microhelix (mihx) on the gel using ImageJ software. Fx: Flexizyme, mihx: microhelix. The red arrows indicate the selected acylation reaction conditions for tRNA acylation with the substrate. 3-DNB and 6-DNB were not charged to mihx, presumably because of poor watersolubility. Substrate structures for 1-7 are shown in the characterization data above. Gel data representative of three independent experiments. The samples derive from the same experiment and the densitometric analysis was performed in parallel on different gels. Supplementary Figure 2. Characterization of the pyridazinone products. The peaks marked as a hash (#) correspond to the theoretical mass of a truncated peptide (strep-tag reporter peptide) which does not contain both γ-keto esters and hydrazine at the N-terminus. The peaks marked as an asterisk (*) correspond to the theoretical mass of peptide that includes a Ser misincorporated at the Thr codon, resulting in a linear product rather than the formation of pyridazinone. Data representative of three independent experiments.   The β-nitrogen atom of hydrazineyl ester (green) coming into the A-site of the ribosome attacks the carbonyl of the ketone of γ-keto ester (orange) to form an imine followed by removal of water. Next, the αnitrogen atom attacks the ester bond to tRNA, thereby resulting in the formation of 2,6-substituted pyridazinone bond. (B) The α-nitrogen atom forms a hydrazone with ketone and then β-nitrogen atom forms a hydrazone, resulting in production of a 2,6-substituted pyridazinone bond. (C) With the similar mechanism, when the a-nitrogen atom of hydrazineyl ester forms hydrazone first and the b-nitrogen atom forms a peptide bond, 1,6-substituted pyridazinone is produced. (D) The b-nitrogen atom forms an amide and then the a-nitrogen atom forms a hydrazone with ketone, resulting in production of a 1,6-substituted pyridazinone.  Figure 8. Monitoring the pyridazinone formation reaction. The pyridazinone was produced in the reaction of cyanomethyl 4-oxo-4-phenylbutanoate with ethyl hydrazinoacetate hydrochloride in EtOH/H2O (3/2: v/v) at 37 °C. The reactions were monitored by LC-MS with different substrate concentrations of 40 μM (A), 4 mM (B), and 40 mM (C). The pyridazinone product was only observed when the concentration of reactants was 1,000 times higher than that used in the ribosome-mediated reaction. The fraction collected at the 5.5 min peak (red box in panel C) was confirmed to be a 2,6substituted pyridazinone (D) through 1 H NMR spectroscopy (400 MHz, CDCl3). Data representative of three independent experiments.
Supplementary Figure 9. Estimated yields of pyridazinone-based peptides. We separately prepared an internal standard peptide (fMetThrTrpSerHisProGlnPheGluLys) using the same plasmid and the natural 20 amino acids in the PURExpress TM . We produced ~1 µg of the standard peptide (PURExpress TM system produces ~100ng per µL of reaction volume) 7 , mixed 100 ng of the peptide standard with each pyridazinone peptide (panel A or B) and analyzed the mixture by MALDI-TOF. After characterization of each peak using the information on panel C, we calculated the relative peak area of the pyridazinone peptides formed between substrates 1 and 5 or 1 and 6. The relative peak area of each peptide containing a pyridazinone is 6.4 and 3.5 %, which indicates ~60 and 30 ng of the target peptides is obtained in a 10 µL reaction. Data representative of three independent experiments. Supplementary Figure 10. Effect of engineered translational machinery on the pyridazinone bond formation with 1 and 5. γKPhe (1) and (S)-HzPhe (5) were charged to tRNA fMet (CAU) and tRNA Pro1E2 (GGU), respectively by Fx and subsequently added to the PURExpress TM system. The 1 and 5 delivered to the ribosome on tRNA fMet (CAU) and tRNA Pro1E2 (GGU), respectively, were consecutively incorporated into a peptide polymer and permitted to undergo water condensation reactions, yielding a pyridazinone bond. (A-B) The pyridazinone bond was produced either in the presence of the wild-type and an engineered ribosome.
(C-D) Peptides containing a pyridazinone at the N-terminus were observed in a low yield when an additional translational machinery, EF-P, was supplemented. Of note, a custom-made Δ (aa, tRNA, ribosome) PURExpress system (NEB, E3315Z) supplying the wild-type ribosome in a separate tube was used for the condensation reactions with engineered ribosomes. The wild-type ribosome supplied in the kit was not used, however, based on previous literature, we expected the engineered 040329 ribosomes to constitute ~25% of the purified ribosome population.